Aerodynamically light particles for pulmonary drug delivery

ABSTRACT

Improved aerodynamically light particles for delivery to the pulmonary system, and methods for their preparation and administration are provided. In a preferred embodiment, the aerodynamically light particles are made of a biodegradable material and have a tap density less than 0.4 g/cm 3  and a mass mean diameter between 5 μm and 30 μm. The particles may be formed of biodegradable mat as biodegradable polymers. For example, the particles may be formed of a functionalized polyester graft copolymer consisting of a linear α-hydroxy-acid polyester backbone having at least one amino acid group incorporated therein and at least one poly(amino acid) side chain extending from an amino acid group in the polyester backbone. In one embodiment, aerodynamically light particles having a large mean diameter, for example greater than 5 μm, can be used for enhanced delivery of a therapeutic or diagnostic agent to the alveolar region of the lung. The aerodynamically light particles optionally can incorporate a therapeutic or diagnostic agent, and may be effectively aerosolized for administration to the respiratory tract to permit systemic or local delivery of wide variety of incrorporated agents.

CROSS REFERENCE TO RELATED APPLICATION

This application is the National Stage Application under 35 U.S.C §371(c) of PCT US97/08895 filed on May 23, 1997 and claiming priority toU.S. application Ser. No. 08/739,308, filed on Oct. 29, 1996, now U.S.Pat. No. 5,874,064, and of U.S. application Ser. No. 08/655,570, filedon May 24, 1996 now abandoned.

BACKGROUND OF THE INVENTION

The present application relates generally to biodegradable particles oflow density and large size for delivery to the pulmonary system.

Biodegradable particles have been developed for the controlled-releaseand delivery of protein and peptide drugs. Langer, R., Science, 249:1527-1533 (1990). Examples include the use of biodegradable particlesfor gene therapy (Mulligan, R. C. Science, 260: 926-932 (1993)) and for‘single-shot’ immunization by vaccine delivery (Eldridge et al., Mol.Iminunol., 28: 287-294 (1991)).

Aerosols for the delivery of therapeutic agents to the respiratory tracthave been developed. Adjei, A. and Garren, J. Pharm. Res. 7, 565-569(1990); and Zanen, P. and Lamm, J. -W. J. Int. J. Pharm. 114, 111-115(1995). The respiratory tract encompasses the upper airways, includingthe oropharynx and larynx, followed by the lower airways, which includethe trachea followed by bifurcations into the bronchi and bronchioli.The upper and lower airways are called the conducting airways. Theterminal bronchioli then divide into respiratory bronchioli which thenlead to the ultimate respiratory zone, the alveoli, or deep lung. Gonda,I. “Aerosols for delivery of therapeutic and diagnostic agents to therespiratory tract,” in Critical Reviews in Therapeutic Drug CarrierSystems 6:273-313, 1990. The deep lung, or alveoli, are the primarytarget of inhaled therapeutic aerosols for systemic drug delivery.

Inhaled aerosols have been used for the treatment of local lungdisorders including asthma and cystic fibrosis (Anderson et al., Am.Rev. Respir. Dis., 140: 1317-1324 (1989)) and have potential for thesystemic delivery of peptides and proteins as well (Patton and Platz,Advanced Drug Delivery Reviews, 8:179-196 (1992)). However, pulmonarydrug delivery strategies present many difficulties for the delivery ofmacromolecules; these include protein denaturation duringaerosolization, excessive loss of inhaled drug in the oropharyngealcavity (often exceeding 80%), poor control over the site of deposition,irreproducibility of therapeutic results owing to variations inbreathing patterns, the often too-rapid absorption of drug potentiallyresulting in local toxic effects, and phagocytosis by lung macrophages.

Considerable attention has been devoted to the design of therapeuticaerosol inhalers to improve the efficiency of inhalation therapies.Timsina et. al., Int. J. Pharm. 101, 1-13 (1995); and Tansey, I. P.,Spray, Technol. Market 4, 26-29 (1994). Attention has also been given tothe design of dry powder aerosol surface texture, regarding particularlythe need to avoid particle aggregation, a phenomenon which considerablydiminishes the efficiency of inhalation therapies. French, D. L.,Edwards, D. A. and Niven, R. W., J. Aerosol Sci. 27, 769-783 (1996).Attention has not been given to the possibility of using large particlesize (greater than 5 μm) as a means to improve aerosolizationefficiency. despite the fact that intraparticle adhesion diminishes withincreasing particle size. French, D. L., Edwards, D. A. and Niven, R. W.J. Aerosol Sci. 27, 769-783 (1996). This is because particles ofstandard mass density (mass density near 1 g/cm³) and mean diametersgreater than 5 μm are known to deposit excessively in the upper airwaysor in the inhaler device. Heyder, J. et al., J. Aerosol Sci., 17:811-825 (1986). For this reason, dry powder aerosols for inhalationtherapy are generally produced with mean diameters primarily in therange of less than 5 μm. Ganderton. D., J. Biopharnaceutical Sciences3:101-105 (1992): and Gonda, I “Physico-Chemical Principles in AerosolDelivery,” in Topics in Pharmaceutical Sciences 1991, Crommelin, D. J.and K. K. Midha, Eds., Medpharm Scientific Publishers, Stuttgart, pp.95-115, 1992. Large “carrier” particles (containing no drug) have beenco-delivered with therapeutic aerosols to aid in achieving efficientaerosolization among other possible benefits. French, D. L., Edwards, D.A. and Niven, R. W. J. Aerosol Sci. 27, 769-783 (1996).

Local and systemic inhalation therapies can often benefit from arelatively slow controlled release of the therapeutic agent. Gonda, I.,“Physico-chemical principles in aerosol delivery,” in: Topics inPharmaceutical Sciences 1991, D. J. A. Crommelin and K. K. Midha, Eds.,Stuttgart: Medpharm Scientific Publishers, pp. 95-117, (1992). Slowrelease from a therapeutic aerosol can prolong the residence of anadministered drug in the airways or acini, and diminish the rate of drugappearance in the bloodstream. Also, patient compliance is increased byreducing the frequency of dosing. Langer, R., Science, 249:1527-1533(1990); and Gonda, I. “Aerosols for delivery of therapeutic anddiagnostic agents to the respiratory tract,” in Critical Reviews inTherapeutic Drug Carrier Svstems 6:273-313, (1990).

The human lungs can remove or rapidly degrade hydrolytically cleavabledeposited aerosols over periods ranging from minutes to hours. In theupper airways, ciliated epithelia contribute to the “mucociliaryescalator” by which particles are swept from the airways toward themouth. Pavia, D. “Lung Mucociliary Clearance,” in Aerosols and the Lung:Clinical and Experimental Aspects, Clarke, S. W. and Pavia, D., Eds.,Butterworths, London, 1984. Anderson et al., Am. Rev. Respir. Dis., 140:1317-1324 (1989). In the deep lungs, alveolar macrophages are capable ofphagocytosing particles soon after their deposition. Warheit, M. B. andHartsky, M. A., Microscopy Res. Tech. 26: 412-422 (1993); Brain, J. D.,“Physiology and Pathophysiology of Pulmonary Macrophages,” in TheReticuloendothelial System, S. M. Reichard and J. Filkins, Eds., Plenum,New York, pp. 315-327, 1985; Dorries. A. M. and Valberg, P. A., Am. Rev.Resp. Disease 146, 831-837 (1991); and Gehr, P. et al. Microscopy Res.and Tech. 26, 423-436 (1993). As the diameter of particles exceeds 3 μm,there is increasingly less phagocytosis by macrophages. Kawaguchi, H. etal., Biomaterials 7: 61-66 (1986); Krenis, L. J. and Strauss, B., Proc.Soc. Exp. Med., 107:748-750 (1961); and Rudt, S. and Muller, R. H., J.Contr. Rel., 22: 263-272 (1992). However, increasing the particle sizealso minimizes the probability of particles (possessing standard massdensity) entering the airways and acini due to excessive deposition inthe oropharyngeal or nasal regions. Heyder, J. et al., J. Aerosol Sci.,17: 811-825 (1986). An effective dry-powder inhalation therapy for bothshort and long term release of therapeutics, either for local orsystemic delivery, requires a powder that displays minimum aggregationand is capable of avoiding or suspending the lung's natural clearancemechanisms until drugs have been effectively delivered.

There is a need for improved inhaled aerosols for pulmonary delivery oftherapeutic agents which are capable of delivering the drug in aneffective amount into the airways or the alveolar zone of the lung.There further is a need for the development of drug carriers for use asinhaled aerosols which are biodegradable and are capable of controlledrelease of drug within the airways or in the alveolar zone of the lung.

It is therefore an object of the present invention to provide improvedcarriers for the pulmonary delivery of therapeutic and diagnosticagents. It is a further object of the invention to provide inhaledaerosols which are effective carriers for delivery of therapeutic ordiagnostic agents to the deep lung. It is another object of theinvention to provide carriers for pulmonary delivery which avoidphagocytosis in the deep lung. It is a further object of the inventionto provide carriers for pulmonary delivery which are capable ofbiodegrading and optionally releasing incorporated agents at acontrolled rate.

SUMMARY OF THE INVENTION

Improved aerodynamically light particles for delivery to the pulmonarysystem, and methods for their preparation and administration areprovided. In a preferred embodiment, the particles are made of abiodegradable material, have a tap density less than 0.4 g/cm³ and amean diameter between 5 μm and 30 μm. In one embodiment, for example, atleast 90% of the particles have a mean diameter between 5 μm and 30 μm.The particles may be formed of biodegradable materials such asbiodegradable synthetic polymers, proteins, or other water-solublematerials such as certain polysaccharides. For example, the particlesmay be formed of a functionalized polyester graft copolymer with alinear α-hydroxy-acid polyester backbone with at least one amino acidresidue incorporated per molecule therein and at least one poly(aminoacid) side chain extending from an amino acid group in the polyesterbackbone. Other examples include particles formed of water-solubleexcipients, such as trehalose or lactose, or proteins, such as lysozymeor insulin. The particles can be used for delivery of a therapeutic ordiagnostic agent to the airways or the alveolar region of the lung. Theparticles may be effectively aerosolized for administration to therespiratory tract and can be used to systemically or locally deliver awide variety of incorporated agents. The particles incorporating anagent can optionally be co-delivered with larger carrier particles, notcarrying an incorporated agent, which have, for example, a mean diameterranging between about 50 μm and 100 μm.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph comparing total particle mass of aerodynamically lightand non-light, control particles deposited on the nonrespirable andrespirable stages of a cascade impactor following aerosolization.

FIG. 2 is a graph comparing total particle mass deposited in the tracheaand after the carina (lungs) in rat lungs and upper airways followingintratracheal aerosolization during forced ventilation ofaerodynamically light poly(lactic acid-co-lysine-graft-lysine)(PLAL-Lys) particles and control, non-light poly(L-lactic acid) (PLA)particles.

FIG. 3 is a graph comparing total particle recovery of aerodynamicallylight PLAL-Lys particles and control PLA particles in rat lungsfollowing bronchoalveolar lavage.

FIG. 4 is a graph representing serum insulin levels (ng/ml) over time(hours) following administration via inhalation or subcutaneousinjection of porous PLGA particles.

FIG. 5 is a graph representing serum insulin levels (ng/ml) over time(hours) following administration via inhalation or subcutaneousinjection of non-porous PLGA particles. Darkened circles representadministration via inhalation. Darkened triangles representadministration via subcutaneous injection. Empty diamonds representuntreated controls.

FIG. 6 is a graph representing serum glucose concentrations (mg/dl)following administration of porous PLGA particles via inhalation.Darkened circles represent administration via inhalation. Darkenedtriangles represent untreated controls.

FIG. 7 is a graph representing serum testosterone levels (ng/ml) overtime (hours) following administration via inhalation or subcutaneousinjection of porous PLGA particles with a diameter of 20.4 μm. Darkenedcircles represent administration via inhalation. Darkened trianglesrepresent administration via subcutaneous injection.

FIG. 8 is a graph representing serum testosterone levels (ng/ml) overtime (hours) following administration via inhalation or subcutaneousinjection of porous PLGA particles with a diameter of 10.1 μm. Darkenedcircles represent administration via inhalation. Darkened trianglesrepresent administration via subcutaneous injection.

DETAILED DESCRIPTION OF THE INVENTION

Aerodynamically light, biodegradable particles for improved delivery tothe respiratory tract are provided. The particles can incorporate atherapeutic or diagnostic agent, and can be used for controlled systemicor local delivery of the agent to the respiratory tract viaaerosolization. In a preferred embodiment, the particles have a tapdensity less than about 0.4 g/cm³. Features of the particle which cancontribute to low tap density include irregular surface texture andporous structure. Administration of the low density particles to thelung by aerosolization permits deep lung delivery of relatively largediameter therapeutic aerosols, for example, greater than 5 μm in meandiameter. A rough surface texture also can reduce particle agglomerationand provide a highly flowable powder, which is ideal for aerosolizationvia dry powder inhaler devices, leading to lower deposition in themouth, throat and inhaler device.

Density and Size of Aerodynamically Light Particles

Particle Size

The mass mean diameter of the particles can be measured using a CoulterCounter. The aerodynamically light particles are preferably at leastabout 5 microns in diameter. The diameter of particles in a sample willrange depending upon depending on factors such as particle compositionand methods of synthesis. The distribution of size of particles in asample can be selected to permit optimal deposition within targetedsites within the respiratory tract.

The particles may be fabricated or separated, for example. byfiltration, to provide a particle sample with a preselected sizedistribution. For example, greater than 30%, 50%, 70%, or 80% of theparticles in a sample can have a diameter within a selected range of atleast 5 μm. The selected range within which a certain percentage of theparticles must fall may be, for example, between about 5 and 30 μm, oroptionally between 5 and 15 μm. In one preferred embodiment, at least aportion of the particles have a diameter between about 9 and 11 μm.Optionally, the particle sample also can be fabricated wherein at least90%, or optionally 95% or 99%, have a diameter within the selectedrange. The presence of the higher proportion of the aerodynamicallylight, larger diameter (at least about 5 μm) particles in the particlesample enhances the delivery of therapeutic or diagnostic agentsincorporated therein to the deep lung.

In one embodiment, in the particle sample, the interquartile range maybe 2 μm, with a mean diameter for example of 7.5, 8.0, 8.5, 9.0, 9.5,10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0 or 13.5 μm. Thus. for example,at least 30%, 40%, 50% or 60% of the particles may have diameters withinthe selected ranges of 5.5-7.5 μm, 6.0-8.0 μm, 6.5-8.5 μm, 7.0-9.0 μm,7.5-9.5 μm, 8.0-10.0 μm, 8.5-10.5 μm, 9.0-11.0 μm, 9.5-11.5 μm,10.0-12.0 μm, 10.5-12.5μm, 11.0-13.0 μm, 11.5-13.5 μm, 12.0-14.0 μm,12.5-14.5 μm or 13.0-15.0 μm. Preferably the above-listed percentages ofparticles have diameters within a 1 μm range, for example, 6.0-7.0 μm,10.0-11.0 μm or 13.0-14.0 μm.

Particles having a tap density less than about 0.4 g/cm³ and a meandiameter of at least about 5 μm are more capable of escaping inertialand gravitational deposition in the oropharyngeal region than smaller ormore dense particles, and are targeted to the airways of the deep lung.The use of larger particles (mean diameter greater than 5 μm) isadvantageous since they are able to aerosolize more efficiently thansmaller, denser particles such as those currently used for inhalationtherapies.

In comparison to smaller, denser particles, the larger (greater than 5μm) aerodynamically light particles also can potentially moresuccessfully avoid phagocytic engulfment by alveolar macrophages andclearance from the lungs, due to size exclusion of the particles. fromthe phagocytes' cytosolic space. For particles of statisticallyisotropic shape (on average, particles of the powder possess nodistinguishable orientation), such as spheres with rough surfaces, theparticle envelope volume is approximately equivalent to the volume ofcytosolic space required within a macrophage for complete particlephagocytosis.

Aerodynamically light particles thus are capable of a longer termrelease of an incorporated diagnostic or therapeutic agent than smaller,denser particles. Following inhalation, aerodynamically lightbiodegradable particles can deposit in the lungs (due to theirrelatively low tap density), and subsequently undergo slow degradationand drug release, without the majority of the particles beingphagocytosed by alveolar macrophages. The agent can be deliveredrelatively slowly into the alveolar fluid, and at a controlled rate intothe blood stream, minimizing possible toxic responses of exposed cellsto an excessively high concentration of the agent. The aerodynamicallylight particles thus are highly suitable for inhalation therapies,particularly in controlled release applications.

The particles may be fabricated with the appropriate material, surfaceroughness, diameter and tap density for localized delivery to selectedregions of the respiratory tract such as the deep lung or upper airways.For example, higher density or larger particles may be used for upperairway delivery, or a mixture of different sized particles in a sample,provided with the same or different incorporated agent may beadministered to target different regions of the lung in oneadministration.

Particle Density and Deposition

The particles have a diameter of at least about 5 μm and optionallyincorporate a therapeutic or diagnostic agent. The particles arepreferably aerodynamically light. As used herein, the phrase“aerodynamically light particles” refers to particles having a tapdensity less than about 0.4 g/cm³. The tap density of particles of a drypowder may be obtained using a Geo Phy™ (Micrometrics Instrument Corp.,Norcross, Ga. 30093). Tap density is a standard measure of the envelopemass density. The envelope mass density of an isotropic particle isdefined as the mass of the particle divided by the minimum sphereenvelope volume within which it can be enclosed.

Inertial impaction and gravitational settling of aerosols arepredominant deposition mechanisms in the airways and acini of the lungsduring normal breathing conditions. Edwards, D. A., J. Aerosol Sci.26:293-317 (1995). The importance of both deposition mechanismsincreases in proportion to the mass of aerosols and not to particle (orenvelope) volume. Since the site of aerosol deposition in the lungs isdetermined by the mass of the aerosol (at least for particles of meanaerodynamic diameter greater than approximately 1 μm), diminishing thetap density by increasing particle surface irregularities and particleporosity permits the delivery of larger particle envelope volumes intothe lungs, all other physical parameters being equal.

The low tap density particles have a small aerodynamic diameter incomparison to the actual envelope sphere diameter. The aerodynamicdiameter, d_(aer), is related to the envelope sphere diameter, d (Gonda,I., “Physico-chemical principles in aerosol delivery,” in Topics inPharmaceutical Sciences 1991 (eds. D. J. A. Crommelin and K. K. Midha),pp. 95-117, Stuttgart: Medpharm Scientific Publishers. 1992) by theformula:

d_(aer)=dρ

where the envelope mass ρ is in units of g/cm³. Maximal deposition ofmonodisperse aerosol particles in the alveolar region of the human lung(approximately 60%) occurs for an aerodynamic diameter of approximatelyd_(aer)=3 μm. Heyder, J. et al., J. Aerosol Sci., 17: 811-825 (1986).Due to their small envelope mass density, the actual diameter d ofaerodynamically light particles comprising a monodisperse inhaled powderthat will exhibit maximum deep-lung deposition is:

d=3/ρμm (where ρ<1 g/cm³);

where d is always greater than 3 μm. For example, aerodynamically lightparticles that display an envelope mass density, ρ=0.1 g/cm³. willexhibit a maximum deposition for particles having envelope diameters aslarge as 9.5 μm. The increased particle size diminishes interparticleadhesion forces. Visser, J., Powder Technology, 58:1-10. Thus, largeparticle size increases efficiency of aerosolization to the deep lungfor particles of low envelope mass density, in addition to contributingto lower phagocytic losses.

Particle Materials

The aerodynamically light particles preferably are biodegradable andbiocompatible, and optionally are capable of biodegrading at acontrolled rate for release of an incorporated thereapeutic ordiagnostic agent. The particles can be made of any material which iscapable of forming a particle having a tap density less than about 0.4g/cm³. Both inorganic and organic materials can be used. Othernon-polymeric materials (e.g. fatty acids) may be used which are capableof forming aerodynamically light particles as defined herein. Differentproperties of the particle can contribute to the aerodynamic lightnessincluding the composition forming the particle, and the presence ofirregular surface structure or pores or cavities within the particle.

Polymeric Particles

The particles may be formed from any biocompatible, and preferablybiodegradable polymer, copolymer, or blend, which is capable of formingparticles having a tap density less than about 0.4 g/cm³.

Surface eroding polymers such as polyanhydrides may be used to form theaerodynamically light particles. For example, polyanhydrides such aspoly[(ρ-carboxyphenoxy)-hexane anhydride] (PCPH) may be used.Biodegradable polyanhydrides are described, for example, in U.S. Pat.No. 4,857,311.

In another embodiment, bulk eroding polymers such as those based onpolyesters, including poly(hydroxy acids), can be used. Preferredpoly(hydroxy acids) are polyglycolic acid (PGA), polylactic acid (PLA)and copolymers and coblends thereof. In one embodiment, the polyesterhas incorporated therein a charged or functionalizable group such as anamino acid.

Other polymers include polyamides, polycarbonates, polyalkylenes such aspolyethylene, polypropylene, poly(ethylene glycol), poly(ethyleneoxide), poly(ethylene terephthalate), poly vinyl compounds such aspolyvinyl alcohols, polyvinyl ethers, and polyvinyl esters, polymers ofacrylic and methacrylic acids, celluloses and other polysaccharides, andpeptides or proteins, or copolymers or blends thereof. Polymers may beselected with or modified to have the appropriate stability anddegradation rates in vivo for different controlled drug deliveryapplications.

Polyester Graft Copolymers

In one preferred embodiment, the aerodynamically light particles areformed from functionalized polyester graft copolymers, as described inHrkach et al., Macromolecules, 28:4736-4739 (1995); and Hrkach et al.,“Poly(L-Lactic acid-co-amino acid) Graft Copolymers: A Class ofFunctional Biodegradable Biomaterials” in Hydrogels and BiodegradablePolymers for Bioapplications, ACS Symposium Series No. 627, Raphael M.Ottenbrite et al., Eds., μmerican Chemical Society, Chapter 8, pp.93-101, 1996. The functionalized graft copolymers are copolymers ofpolyesters, such as poly(glycolic acid) or poly(lactic acid), andanother polymer including functionalizable or ionizable groups, such asa poly(amino acid). In a preferred embodiment, comb-like graftcopolymers are used which include a linear polyester backbone havingamino acids incorporated therein, and poly(amino acid) side chains whichextend from the amino acid residues in the polyester backbone. Thepolyesters may be polymers of α-hydroxy acids such as lactic acid,glycolic acid, hydroxybutyric acid and hydroxyvaleric acid, orderivatives or combinations thereof. The polymers can include ionizableside chains, such as polylysine and polyaniline. Other ionizable groups,such as amino or carboxyl groups, may be incorporated into the polymer.covalently or noncovalently, to enhance surface roughness and porosity.

An exemplary polyester graft copolymer is poly(lacticacid-colysine-graft-lysine) (PLAL-Lys), which has a polyester backboneconsisting of poly(L-lactic acid-co- L-lysine) (PLAL), and graftedpolylysine chains. PLAL-Lys is a comb-like graft copolymer having abackbone composition, for example, of 98 mol % lactic acid and 2 mol %lysine and poly(lysine) side chains extending from the lysine sites ofthe backbone.

PLAL-Lys may be synthesized as follows. First, the PLAL copolymerconsisting of L-lactic acid units and approximately 1-2% N εcarbobenzoxy-L-lysine (Z-L-lysine) units is synthesized as described inBarrera et al., J. Am. Chem. Soc., 115:11010 (1993). Removal of the Zprotecting groups of the randomly incorporated lysine groups in thepolymer chain of PLAL yields the free E-amine which can undergo furtherchemical modification. The use of the poly(lactic acid) copolymer isadvantageous since it biodegrades into lactic acid and lysine, which canbe processed by the body. The existing backbone lysine groups are usedas initiating sites for the growth of poly(amino acid) side chains.

The lysine ε-amino groups of linear poly(L-lactic acid-co-L-lysine)copolymers initiate the ring opening polymerization of an amino acid N-εcarboxyanhydride (NCA) to produce poly(L-lactic acid-co-amino acid)comb-like graft copolymers. In a preferred embodiment, NCAs aresynthesized by reacting the appropriate amino acid with triphosgene.Daly et al., Tetrahedron Lett., 29:5859 (1988). The advantage of usingtriphosgene over phosgene gas is that it is a solid material, andtherefore, safer and easier to handle. It also is soluble in THF andhexane so any excess is efficiently separated from the NCAs.

The ring opening polymerization of amino acid N-carboxyanhydrides (NCAs)is initiated by nucleophilic initiators such as amines, alcohols, andwater. The primary amine initiated ring opening polymerization of NCAsallows efficient control over the degree of polymerization when themonomer to initiator ratio (M/I) is less than 150. Kricheldorf, H. R. inModels of Biopolymers by Ring-Opening Polymerization, Penczek, S., Ed.,CRC Press, Boca Raton, 1990. Chapter 1; Kricheldorf, H. R.α-Aminoacid-N-Carboxy-Anhydrides and Related Heterocycles,Spriner-Verlag, Berlin, 1987; and Imanishi, Y. in Ring-OpeningPolymerization, Ivin, K. J. and Saegusa, T., Eds., Elsevier, London,1984, Volume 2, Chapter 8. Methods for using lysine ε-amino groups aspolymeric initiators for NCA polymerizations are described in the art.Sela, M. et al., J. Am. Chem. Soc., 78: 746 (1956).

In the reaction of an amino acid NCA with PLAL, the nucleophilic primaryε-amino group of the lysine side chain attacks C-5 of the NCA. Thisleads to ring opening to form an amide linkage, accompanied by evolutionof a molecule of CO₂. The amino group formed by the evolution of CO₂propogates the polymerization by attacking subsequent NCA molecules. Thedegree of polymerization of the poly(amino acid) side chains, the aminoacid content in the resulting graft copolymers and the physical andchemical characteristics of the resulting copolymers can be controlledby adjusting the ratio of NCA to lysine ε-amino groups in the PLALpolymer, for example, by adjusting the length of the poly(amino acid)side chains and the total amino acid content.

The poly(amino acid) side chains grafted onto or incorporated into thepolyester backbone can include any amino acid. such as aspartic acid,alanine or lysine, or mixtures thereof. The functional groups present inthe amino acid side chains, which can be chemically modified, includeamino, carboxylic acid, thiol, guanido, imidazole and hydroxyl groups.As used herein, the term “amino acid” includes natural and syntheticamino acids and derivatives thereof. The polymers can be prepared with arange of side chain lengths. The side chains preferably include between10 and 100 amino acids, and have an overall amino acid content between 7and 72%. However, the side chains can include more than 100 amino acidsand can have an overall amino acid content greater than 72%, dependingon the reaction conditions. Poly(amino acids) can be grafted to the PLALbackbone in any suitable solvent. Suitable solvents include polarorganic solvents such as dioxane, DMF, CH₂Cl₂, and mixtures thereof. Ina preferred embodiment, the reaction is conducted in dioxane at roomtemperature for a period of time between about 2 and 4 days.

Alternatively, the particles may be formed from polymers or blends ofpolymers with different polyester/amino acid backbones and grafted aminoacid side chains. For example, poly(lacticacid-co-lysine-graft-alanine-lysine) (PLAL-Ala-Lys), or a blend ofPLAL-Lys with poly(lactic acid-co-glycolic acid-block-ethylene oxide)(PLGA-PEG) (PLAL-Lys-PLGA-PEG) may be used.

In the synthesis, the graft copolymers may be tailored to optimizedifferent characteristics of the aerodynamically light particleincluding: i) interactions between the agent to be delivered and thecopolymer to provide stabilization of the agent and retention ofactivity upon delivery; ii) rate of polymer degradation and, thereby,rate of drug release profiles; iii) surface characteristics andtargeting capabilities via chemical modification; and iv) particleporosity.

Therapeutic Agents

Any of a variety of therapeutic agents can be incorporated within theparticles, which can locally or systemically deliver the incorporatedagents following administration to the lungs of an animal. Examplesinclude synthetic inorganic and organic compounds or molecules, proteinsand peptides, polysaccharides and other sugars, lipids, and nucleic acidmolecules having therapeutic, prophylactic or diagnostic activities.Nucleic acid molecules include genes, antisense molecules which bind tocomplementary DNA to inhibit transcription, ribozymes and ribozyme guidesequences. The agents to be incorporated can have a variety ofbiological activities, such as vasoactive agents, neuroactive agents,hormones, anticoagulants, immunomodulating agents, cytotoxic agents,prophylactic agents, antibiotics, antivirals, antisense, antigens, andantibodies. In some instances, the proteins may be antibodies orantigens which otherwise would have to be administered by injection toelicit an appropriate response. Compounds with a wide range of molecularweight, for example, between 100 and 500,000 grams per mole, can beencapsulated.

Proteins are defined as consisting of 100 amino acid residues or more;peptides are less than 100 amino acid residues. Unless otherwise stated,the term protein refers to both proteins and peptides. Examples includeinsulin and other hormones. Polysaccharides, such as heparin, can alsobe administered.

Aerosols including the aerodynamically light particles are useful for avariety of inhalation therapies. The particles can incorporate small andlarge drugs, release the incorporated drugs over time periods rangingfrom hours to months, and withstand extreme conditions duringaerosolization or following deposition in the lungs that might otherwiseharm the encapsulated agents.

The agents can be locally delivered within the lung or can besystemically administered. For example, genes for the treatment ofdiseases such as cystic fibrosis can be administered, as can betaagonists for asthma. Other specific therapeutic agents include insulin,calcitonin, leuprolide (or LHRH), G-CSF, parathyroid hormone-relatedpeptide, somatostatin, testosterone, progesterone, estradiol, nicotine,fentanyl, norethisterone, clonidine, scopolomine, salicylate, cromolynsodium, salmeterol, formeterol, albeterol, and valium.

Diagnostic Agents

Any of a variety of diagnostic agents can be incorporated within theparticles, which can locally or systemically deliver the incorporatedagents following administration to the lungs of an animal, includinggases and other imaging agents.

Gases

Any biocompatible or pharmacologically acceptable gas can beincorporated into the particles or trapped in the pores of theparticles. The term gas refers to any compound which is a gas or capableof forming a gas at the temperature at which imaging is being performed.The gas may be composed of a single compound such as oxygen, nitrogen,xenon, argon, nitrogen or a mixture of compounds such as air. Examplesof fluorinated gases include CF₄, C₂F₆, C₃F₈, C₄F₈, SF₆, C₂F₄, and C₃F₆.

Other Imaging Agents

Other imaging agents which may be utilized include commerciallyavailable agents used in positron emission tomography (PET), computerassisted tomography (CAT), single photon emission computerizedtomography, x-ray, fluoroscopy, and magnetic resonance imaging (MRI).

Examples of suitable materials for use as contrast agents in MRI includethe gatalinium chelates currently available, such as diethylene triaminepentacetic acid (DTPA) and gatopentotate dimeglumine, as well as iron,magnesium, manganese, copper and chromium.

Examples of materials useful for CAT and x-rays include iodine basedmaterials for intravenous administration, such as ionic monomerstypified by diatrizoate and iothalamate, non-ionic monomers such asiopamidol, isohexol, and ioversol, non-ionic dimers, such as iotrol andiodixanol, and ionic dimers, for example, ioxagalte.

Particles incorporating these agents can be detected using standardtechniques available in the art and commercially available equipment.

Formation of Aerodynamically Light Polymeric Particles

Aerodynamically light polymeric particles may be prepared using singleand double emulsion solvent evaporation, spray drying, solventextraction or other methods well known to those of ordinary skill in theart. The particles may be made, for example, using methods for makingmicrospheres or microcapsules known in the art.

Methods for making microspheres are described in the literature, forexample, in Mathiowitz and Langer, J. Controlled Release 5,13-22 (1987);Mathiowitz et al., Reactive Polymers 6, 275-283 (1987); and Mathiowitzet al., J. Appl. Polymer Sci. 35, 755-774 (1988). The selection of themethod depends on the polymer selection, the size, external morphology,and crystallinity that is desired, as described, for example, byMathiowitz et al., Scanning Microscopy 4,329-340 (1990); Mathiowitz etal., J. Appl. Polymer Sci. 45, 125-134 (1992); and Benita et al., J.Pharm. Sci. 73, 1721-1724 (1984).

In solvent evaporation, described for example, in Mathiowitz, et al.,(1990), Benita, and U.S. Pat. No. 4,272,398 to Jaffe, a polymer isdissolved in a volatile organic solvent, such as methylene chloride.Several different polymer concentrations can be used, for example,between 0.05 and 0.20 g/ml. An agent to be incorporated, either insoluble form or dispersed as fine particles, is optionally added to thepolymer solution, and the mixture is suspended in an aqueous phase thatcontains a surface active agent such as poly(vinyl alcohol). Theresulting emulsion is stirred until most of the organic solventevaporates, leaving solid microspheres, which may be washed with waterand dried overnight in a lyophilizer.

Microspheres with different sizes (typically between 1 and 1000 microns)and morphologies can be obtained. This method is especially useful forrelatively stable polymers such as polyesters and polystyrene. However,labile polymers such as polyanhydrides may degrade due to exposure towater. Solvent removal may be a preferred method for preparingmicrospheres from these polymers.

Solvent removal was primarily designed for use with polyanhydrides. Inthis method, a therapeutic or diagnostic agent can be dispersed ordissolved in a solution of a selected polymer in a volatile organicsolvent like methylene chloride. The mixture can then be suspended inoil, such as silicon oil, by stirring, to form an emulsion. As thesolvent diffuses into the oil phase, the emulsion droplets harden intosolid polymer microspheres. Unlike solvent evaporation, this method canbe used to make microspheres from polymers with high melting points anda wide range of molecular weights. Microspheres having a diameterbetween one and 300 microns can be obtained using this procedure.

Targeting of Particles

Targeting molecules can be attached to the particles via reactivefunctional groups on the particles. For example, targeting molecules canbe attached to the amino acid groups of functionalized polyester graftcopolymer particles, such as PLAL-Lys particles. Targeting moleculespermit binding interactions of the particle with specific receptorsites, such as those within the lungs. The particles can be targeted byattaching ligands which specifically or non-specifically bind toparticular targets. Exemplary targeting molecules include antibodies andfragments thereof including the variable regions, lectins, and hormonesor other organic molecules capable of specific binding to receptors onthe surfaces of the target cells.

Administration

The particles can be administered to the respiratory system alone or inany appropriate pharmaceutically acceptable carrier, such as a liquid,for example saline, or a powder. In one embodiment, particlesincorporating a prophylactic, therapeutic or diagnostic agent arecodelivered with larger carrier particles that do not include anincorporated agent. Preferably, the larger particles have a mass meandiameter between about 50 and 100 μm.

Aerosol dosage, formulations and delivery systems may be selected for aparticular therapeutic application, as described, for example, in Gonda,I. “Aerosols for delivery of therapeutic and diagnostic agents to therespiratory tract,” in Critical Reviews in Therapeutic Drug CarrierSystems, 6:273-313, 1990; and in Moren, “Aerosol dosage forms andformulations,” in: Aerosols in Medicine. Principles, Diagnosis andTherapy, Moren, et al., Eds, Elsevier, Amsterdam, 1985.

The greater efficiency of aerosolization by aerodynamically lightparticles of relatively large size permits more of an incorporated agentto be delivered than is possible with the same mass of relatively denseaerosols. The relatively large particle size also minimizes potentialdrug losses caused by particle phagocytosis. When the particles areformed from biocompatible polymers, the system can provide controlledrelease in the lungs and long-time local action or systemicbioavailability of the incorporated agent. Denaturation ofmacromolecular drugs can be minimized during aerosolization sincemacromolecules are contained and protected within a polymeric shell. Theenzymatic degradation of proteins or peptides can be minimized byco-incorporating peptidase-inhibitors.

Diagnostic Applications

The particles can be combined with a pharmaceutically acceptablecarrier, then an effective amount for detection administered to apatient via inhalation. Particles containing an incorporated imagingagent may be used for a variety of diagnostic applications, includingdetecting and characterizing tumor masses and tissues.

The present invention will be further understood by reference to thefollowing non-limiting examples.

EXAMPLE 1 Synthesis of Aerodynamically LightPoly[(p-carboxyphenoxy)-hexane anhydride] (“PCPH”) Particles

Aerodynamically light poly[(p-carboxyphenoxy)-hexane anhydride] (“PCPH”)particles were synthesized as follows. 100 mg PCPH (MW approximately25,000) was dissolved in 3.0 mL methylene chloride. To this clearsolution was added 5.0 mL 1% w/v aqueous polyvinyl alcohol (PVA, MWapproximately 25,000, 88 mole % hydrolyzed) saturated with methylenechloride, and the mixture was vortexed (Vortex Genie 2, FisherScientific) at maximum speed for one minute. The resulting milky-whiteemulsion was poured into a beaker containing 95 mL 1% PVA andhomogenized (Silverson Homogenizers) at 6000 RPM for one minute using a0.75 inch tip. After homogenization, the mixture was stirred with amagnetic stirring bar and the methylene chloride quickly extracted fromthe polymer particles by adding 2 mL isopropyl alcohol. The mixture wasstirred for 35 minutes to allow complete hardening of themicroparticles. The hardened particles were collected by centrifugationand washed several times with double distilled water. The particles werefreeze dried to obtain a free-flowing powder void of clumps. Yield,85-90%.

The mean diameter of this batch was 6.0 μm, however, particles with meandiameters ranging from a few hundred nanometers to several millimetersmay be made with only slight modifications. Scanning electron micrographphotos of a typical batch of PCPH particles showed the particles to behighly porous with irregular surface shape. The particles had a tapdensity less than 0.4 g/cm³.

EXAMPLE 2 Synthesis of PLAL-Lys and PLAL-Lys-Ala Polymeric andCopolymeric Particles

Aerodynamically Light PLAL-Lys Particles

PLAL-Lys particles were prepared by dissolving 50 mg of the graftcopolymer in 0.5 ml dimethylsulfoxide, then adding 1.5 mldichloromethane dropwise. The polymer solution is emulsified in 100 mlof 5% w/v polyvinyl alcohol solution (average molecular weight 25KDa,88% hydrolyzed) using a homogenizer (Silverson) at a speed ofapproximately 7500 rpm. The resulting dispersion was stirred using amagnetic stirrer for 1 hour. Following this period, the pH was broughtto between 7.0 and 7.2 by addition of a 0.1 N NaOH solution. Stirringwas continued for an additional 2 hours until the dichloromethane wascompletely evaporated and the particles hardened. The particles werethen isolated by centrifugation at 4000 rpm (1600 g) for 10 minutes(Sorvall RC-5 B). The supernatant was discarded and the precipitatewashed three times with distilled water followed by centrifugation for10 minutes at 4000 rpm each time. Finally, the particles wereresuspended in 5 ml of distilled water, the dispersion frozen in liquidnitrogen, and lyophilized (Labconco freeze dryer 8) for at least 48hours. Particle sizing was performed using a Coulter counter. Averageparticle mean diameters ranged from between 100 nm and 14 μm, dependingupon processing parameters such as homogenization speed and time. Allparticles exhibited tap densities less than 0.4 g/cm³. Scanning electronmicrograph photos of the particles showed them to be highly porous withirregular surfaces.

Aerodynamically Light PLAL-Ala-Lys Particles

100 mg of PLAL-Ala-Lys was completely dissolved in 0.4 mltrifluoroethanol, then 1.0 ml methylene chloride was added dropwise. Thepolymer solution was emulsified in 100 ml of 1% w/v polyvinyl alcoholsolution (average molecular weight 25 KDa, 80% hydrolyzed) using asonicator (Sonic&Material VC-250) for 15 seconds at an output of 40 W. 2ml of 1% PVA solution was added to the mixture and it was vortexed atthe highest speed for 30 seconds. The mixture was quickly poured into abeaker containing 100 ml 0.3% PVA solution, and stirred for three hoursallowing evaporation of the methylene chloride. Scanning electronmicrograph photos of the particles showed them to possess highlyirregular surfaces.

Aerodynamically Light Copolymer Particles

Polymeric aerodynamically light particles consisting of a blend ofPLAL-Lys and PLGA-PEG were made. 50 mg of the PLGA-PEG polymer(molecular weight of PEG: 20 KDa, 1:2 weight ratio of PEG:PLGA, 75:25lactide:glycolide) was completely dissolved in 1 ml dichloromethane. 3mg of poly(lactide-co-lysine)-polylysine graft copolymer was dissolvedin 0.1 ml dimethylsulfoxide and mixed with the first polymer solution.0.2 ml of TE buffer, pH 7.6, was emulsified in the polymer solution byprobe sonication (Sonic&Material VC-250) for 10 seconds at an output of40W. To this first emulsion, 2 ml of distilled water was added and mixedusing a vortex mixer at 4000 rpm for 60 seconds. The resultingdispersion was agitated by using a magnetic stirrer for 3 hours untilmethylene chloride was completely evaporated and microspheres formed.The spheres were then isolated by centrifugation at 5000 rpm for 30 min.The supernatant was discarded, the precipitate washed three times withdistilled water and resuspended in 5 ml of water. The dispersion wasfrozen in liquid nitrogen and lyophilized for 48 hours.

By scanning electron microscopy (SEM), the PLAL-Lys-PLGAPEG particleswere highly surface rough and porous. The particles had a mean particlediameter of 7 μm. The blend of PLAL-Lys with poly(lactic acid) (PLA)and/or PLGA-PEG copolymers can be adjusted to adjust particle porosityand size.

Variables which may be manipulated to alter the size distribution of theparticles include: polymer concentration, polymer molecular weight,surfactant type (e.g., PVA, PEG, etc.), surfactant concentration, andmixing intensity. Variables which may be manipulated to alter thesurface shape and porosity of the particles include: polymerconcentration, polymer molecular weight, rate of methylene chlorideextraction by isopropyl alcohol (or another miscible solvent), volume ofisopropyl alcohol added, inclusion of an inner water phase, volume ofinner water phase, inclusion of salts or other highly water-solublemolecules in the inner water phase which leak out of the hardeningsphere by osmotic pressure, causing the formation of channels, or pores,in proportion to their concentration, and surfactant type andconcentration.

Additionally, processing parameters such as homogenization speed andtime can be adjusted. Neither PLAL, PLA nor PLGA-PEG alone yields anaerodynamically light structure when prepared by these techniques.

EXAMPLE 3 Synthesis of Spray-Dried Particles

Aerodynamically Light Particles Containing Polymer and Drug Soluble inCommon Solvent

Aerodynamically light 50:50 PLGA particles were prepared by spray dryingwith testosterone encapsulated within the particles according to thefollowing procedures. 2.0 g poly (D,L-lactic-co-glycolic acid) with amolar ratio of 50:50 (PLGA 50:50, Resomer RG503, B.I. Chemicals,Montvale, N.J.) and 0.50 g testosterone (Sigma Chemical Co., St. Louis,Mo.) were completely dissolved in 100 mL dichloromethane at roomtemperature. The mixture was subsequently spray-dried through a 0.5 mmnozzle at a flow rate of 5 mL/min using a Buchi laboratory spraydrier(model 190, Buchi, Germany). The flow rate of compressed air was 700nl/h. The inlet temperature was set to 30° C. and the outlet temperatureto 25° C. The aspirator was set to achieve a vacuum of −20 to −25 bar.The yield was 51% and the mean particle size was approximately 5 μm. Theparticles were aerodynamically light, as determined by a tap densityless than or equal to 0.4 g/cm³.

Larger particle size can be achieved by lowering the inlet compressedair flow rate, as well as by changing other variables. Porosity andsurface roughness can be increased by varying the inlet and outlettemperatures, among other factors.

Aerodynamically Light Particles Containing Polymer and Drug in DifferentSolvents

Aerodynamically light PLA particles with a model hydrophilic drug(dextran) were prepared by spray drying using the following procedure.2.0 mL of an aqueous 10% w/v FITC-dextran (MW 70,000, Sigma ChemicalCo.) solution was emulsified into 100 mL of a 2% w/v solution of poly(D,L-lactic acid) (PLA, Resomer R206, B.I. Chemicals) in dichloromethaneby probe sonication (Vibracell Sonicator, Branson). The emulsion wassubsequently spray-dried at a flow rate of 5 mL/min with an air flowrate of 700 nl/h (inlet temperature=30° C., outlet temperature=21° C.,−20 mbar vacuum). The yield is 56%. The particles were aerodynamicallylight (tap density less than 0.4 g/cm³).

Aerodynamically Light Protein Particles

Aerodynamically light lysozyme particles were prepared by spray dryingusing the following procedure. 4.75 g lysozyme (Sigma) was dissolved in95 mL double distilled water (5% w/v solution) and spray-dried using a0.5 mm nozzle and a Buchi laboratory spray-drier. The flow rate ofcompressed air was 725 nl/h. The flow rate of the lysozyme solution wasset such that, at a set inlet temperature of 97-100° C., the outlettemperature is between 55 and 57° C. The aspirator was set to achieve avacuum of −30 mbar. The enzymatic activity of lysozyme was found to beunaffected by this process and the yield of the aerodynamically lightparticles (tap density less than 0.4 g/cm³) was 66%.

Aerodynamically Light High-Molecular Weight Water-Soluble Particles

Aerodynamically light dextran particles were prepared by spray dryingusing the following procedure. 6.04 g DEAE dextran (Sigma) was dissolvedin 242 mL double distilled water (2.5% w/v solution) and spray-driedusing a 0.5 mm nozzle and a Buchi laboratory spray-drier. The flow rateof compressed air was 750 nl/h. The flow rate of the DEAE-dextransolution was set such that, at a set inlet temperature of 155° C., theoutlet temperature was 80° C. The aspirator was set to achieve a vacuumof −20 mbar. The yield of the aerodynamically light particles (tapdensity less than 0.4 g/cm³) was 66% and the size range ranged between 1and 15 μm.

Aerodynamically Light Low-Molecular Weight Water-Soluble Particles

Aerodynamically light trehalose particles were prepared by spray dryingusing the following procedure. 4.9 g trehalose (Sigma) was dissolved in192 mL double distilled water (2.5% wlv solution) and spray-dried usinga 0.5 mm nozzle and a Buchi laboratory spray-drier. The flow rate ofcompressed air 650 nl/h. The flow rate of the trehalose solution was setsuch that, at a set inlet temperature of 100° C., the outlet temperaturewas 60° C. The aspirator was set to achieve a vacuum of -30 mbar. Theyield of the aerodynamically light particles (tap density less than 0.4g/cm³) was 36% and the size range ranged between 1 and 15 μm.

Aerodynamically Light Low-Molecular Weight Water-Soluble Particles

Polyethylene glycol (PEG) is a water-soluble macromolecule, however, itcannot be spray dried from an aqueous solution since it melts at roomtemperatures below that needed to evaporate water. PEG was spray-driedat low temperatures from a solution in dichloromethane, a low boilingorganic solvent. Aerodynamically light PEG particles were prepared byspray drying using the following procedure. 5.0 g PEG (MW 15,000-20,000,Sigma) was dissolved in 100 mL double distilled water (5.0% w/vsolution) and spray-dried using a 0.5 mm nozzle and a Buchi laboratoryspray-drier. The flow rate of compressed air was 750 nl/h. The flow rateof the PEG solution was set such that, at a set inlet temperature of 45°C., the outlet temperature was 34-35° C. The aspirator was set toachieve a vacuum of −22 mbar. The yield of the aerodynamically lightparticles (tap density less than 0.4 g/cm³) was 67% and the size rangeranged between 1 and 15 μm.

EXAMPLE 4 Rhodamine Isothiocyanate Labeling of PLAL and PLAL-LysParticles

Aerodynamically light particles were compared with control particles,referred to herein as “non-light” particles. Lysine amine groups on thesurface of aerodynamically light (PLAL-Lys) and control, non-light(PLAL) particles, with similar mean diameters (between 6 and 7 μm) andsize distributions (standard deviations between 3 and 4 μm) were labeledwith Rhodamine isothiocyanate. The tap density of the porous PLAL-Lysparticles was 0.1 g/cm³ and that of the denser PLAL particles was 0.8g/cm³.

The rhodamine-labeled particles were characterized by confocalmicroscopy. A limited number of lysine functionalities on the surface ofthe solid particle were able to react with rhodamine isothiocyanate, asevidenced by the fluorescent image. In the aerodynamically lightparticle, the higher lysine content in the graft copolymer and theporous particle structure result in a higher level of rhodamineattachment, with rhodamine attachment dispersed throughout theinterstices of the porous structure. This also demonstrates thattargeting molecules can be attached to the aerodynamically lightparticles for interaction with specific receptor sites within the lungsvia chemical attachment of appropriate targeting agents to the particlesurface.

EXAMPLE 5 Aerosolization of PLAL and PLAL-Lys Particles

To determine whether large aerodynamically light particles can escape(mouth, throat and inhaler) deposition and more efficiently enter theairways and acini than nonporous particles of similar size (referred toherein as non-light or control particles), the aerosolization anddeposition of aerodynamically light PLAL-Lys (mean diameter 6.3 μm) andcontrol, non-light PLAL (mean diameter 6.9 μm) particles was compared invitro using a cascade impactor system.

20 mg of the aerodynamically light or non-light microparticles wereplaced in gelatine capsules (Eli Lilly), the capsules loaded into aSpinhaler dry powder inhaler (DPI) (Fisons), and the DPI activated.Particles were aerosolized into a Mark I Andersen Impactor (AndersenSamplers, Ga.) from the DPI for 30 seconds at 28.3 l/min flow rate. Eachplate of the Andersen Impactor was previously coated with Tween 80 byimmersing the plates in an acetone solution (5% w/vol) and subsequentlyevaporating the acetone in a oven at 60° C. for 5 min. Afteraerosolization and deposition, particles were collected from each stageof the impactor system in separate volumetric flasks by rinsing eachstage with a NaOH solution (0.2 N) in order to completely degrade thepolymers. After incubation at 37° C. for 12 h, the fluorescence of eachsolution was measured (wavelengths of 554 nm excitation, 574 nmemission).

Particles were determined as nonrespirable (mean aerodynamic diameterexceeding 4.7 μm: impactor estimate) if they deposited on the firstthree stages of the impactor, and respirable (mean aerodynamic diameter4.7 μm or less) if they deposited on subsequent stages. FIG. 1 showsthat less than 10% of the non-light (PLAL) particles that exit the DPIare respirable. This is consistent with the large size of themicroparticles and their standard mass density. On the other hand,greater than 55% of the aerodynamically light (PLAL-Lys) particles arerespirable, even though the geometrical dimensions of the two particletypes are almost identical. The lower tap density of the aerodynamicallylight (PLAL-Lys) microparticles is responsible for this improvement inparticle penetration, as discussed further below.

The non-light (PLAL) particles also inefficiently aerosolize from theDPI; typically, less than 40% of the non-light particles exited theSpinhaler DPI for the protocol used. The aerodynamically light(PLAL-Lys) particles exhibited much more efficient aerosolization(approximately 80% of the aerodynamically light microparticles typicallyexited the DPI during aerosolization).

The combined effects of efficient aerosolization and high respirablefraction of aerosolized particle mass means that a far greater fractionof an aerodynamically light particle powder is likely to deposit in thelungs than of a non-light particle powder.

EXAMPLE 6 In Vivo Aerosolization of PLAL and PLAL-Lys Particles

The penetration of aerodynamically light and non-light polymericPLAL-Lys and PLAL microparticles into the lungs was evaluated in an invivo experiment involving the aerosolization of the microparticles intothe airways of live rats.

Male Spraque Dawley rats (150-200 g) were anesthetized using ketamine(90 mg/kg)/xylazine (10 mg/kg). The anesthetized rat was placed ventralside up on a surgical table provided with a temperature controlled padto maintain physiological temperature. The animal was cannulated abovethe carina with an endotracheal tube connected to a Harvard ventilator.The animal was force ventilated for 20 minutes at 300 ml/min. 50 mg ofaerodynamically light (PLAL-Lys) or non-light (PLA) microparticles wereintroduced into the endotracheal tube.

Following the period of forced ventilation, the animal was euthanizedand the lungs and trachea were separately washed using bronchoalveolarlavage. A tracheal cannula was inserted, tied into place, and theairways were washed with 10 ml aliquots of HBSS. The lavage procedurewas repeated until a total volume of 30 ml was collected. The lavagefluid was centrifuged (400 g) and the pellets collected and resuspendedin 2 ml of phenol red-free Hanks balanced salt solution (Gibco, GrandIsland, N.Y.) without Ca² and Mg²⁺ (HBSS). 100 ml were removed forparticle counting using a hemacytometer. The remaining solution wasmixed with 10 ml of 0.4 N NaOH. After incubation at 37° C. for 12 h, thefluorescence of each solution was measured (wavelengths of 554 nmexcitation, 574 nm emission). FIG. 2 is a bar graph showing totalparticle mass deposited in the trachea and after the carina (lungs) inrat lungs and upper airways following intratracheal aerosolizationduring forced ventilation. The PLAL-Lys aerodynamically light particleshad a mean diameter of 6.9 μm. The non-light PLAL particles had a meandiameter of 6.7 μm. Percent tracheal aerodynamically light particledeposition was 54.5, and non-light deposition was 77.0. Percentaerodynamically light particle deposition in the lungs was 46.8 andnon-light deposition was 23.0.

The non-light (PLAL) particles deposited primarily in the trachea(approximately 79% of all particle mass that entered the trachea). Thisresult is similar to the in vitro performance of the non-lightmicroparticles and is consistent with the relatively large size of thenonlight particles. Approximately 54% of the aerodynamically light(PLAL-Lys) particle mass deposited in the trachea. Therefore, about halfof the aerodynamically light particle mass that enters the tracheatraverses through the trachea and into the airways and acini of the ratlungs, demonstrating the effective penetration of the aerodynamicallylight particles into the lungs.

Following bronchoalveolar lavage, particles remaining in the rat lungswere obtained by careful dissection of the individual lobes of thelungs. The lobes were placed in separate petri dishes containing 5 ml ofHBSS. Each lobe was teased through 60 mesh screen to dissociate thetissue and was then filtered through cotton gauze to remove tissuedebris and connective tissue. The petri dish and gauze were washed withan additional 15 ml of HBSS to maximize microparticle collection. Eachtissue preparation was centrifuged and resuspended in 2 ml of HBSS andthe number of particles counted in a hemacytometer. The particle numbersremaining in the lungs following the bronchoalveolar lavage are shown inFIG. 3. Lobe numbers correspond to: 1) left lung, 2) anterior, 3)median, 4) posterior, 5) postcaval. A considerably greater number ofaerodynamically light PLAL-Lys particles enters every lobe of the lungsthan the nonlight PLAL particles, even though the geometrical dimensionsof the two types of particles are essentially the same. These resultsreflect both the efficiency of aerodynamically light particleaerosolization and the propensity of the aerodynamically light particlesto escape deposition prior to the carina or first bifurcation.

EXAMPLE 7 In Vivo Aerosolization of PLGA Porous and Non-Porous ParticlesIncluding Insulin

Insulin was encapsulated into porous and nonporous polymeric particlesto test whether large particle size can increase systemicbioavailability. The mass densities and mean diameters of the twoparticles were designed such that they each possessed an aerodynamicdiameter (approximately 2 μm) suitable for deep lung deposition, withthe mean diameter of the porous particles >5 μm and that of thenonporous particles less than 5 μm (see FIGS. 4-6). Identical masses ofthe porous or nonporous particles were administered to rats as aninhalation aerosol or injected subcutaneously (controls).

Rats were anesthetized and cannulated as previously described. Theanimal was force ventilated for between 10 and 20 minutes at 300 ml/min.Two types of aerosols were delivered to the animal via the endotrachealtube. Following the period of forced ventilation, the neck of the animalwas sutured and the animal revived within one to two hours. Bloodsamples (300 μl) were periodically withdrawn from the tail vein over aperiod of two to six days. These samples were mixed with assay buffer,centrifuged, and the supernatant examined for the presence of(endogenous and exogenous) insulin or testosterone usingradioimmunoassays (ICN Pharmaceuticals, Costa Mesa, Calif.). Glucose wasmeasured using a colorimetric assay (Sigma). Control studies involvedsubcutaneous injection of the same amount of powder as was inhaled. Theparticles were injected into the scruff of the neck.

Serum insulin concentrations were monitored as a function of timefollowing inhalation or injection. For both porous (FIG. 4) andnonporous (FIG. 5) particles, blood levels of insulin reach high valueswithin the first hour following inhalation. Only in the case of thelarge porous particles do blood levels of insulin remain elevated(p<0.05) beyond 4 h, with a relatively constant insulin releasecontinuing to at least 96 h (0.04<p<0.2).

These results are confirmed by serum glucose values which show fallingglucose levels for the first 10 h after inhalation of the porous insulinparticles, followed by relatively constant low glucose levels for theremainder of the 96 h period, as shown in FIG. 6. In the case of smallnonporous insulin particles, initially suppressed glucose values roseafter 24 h.

Similar biphasic release profiles of macromolecules from PLGA polymershave been reported in the literature (S. Cohen et al. Pharm. Res. 8, 713(1991)). For the large porous particles, insulin bioavailabilityrelative to subcutaneous injection is 87.5%, whereas the small nonporousparticles yield a relative bioavailability of 12% following inhalation.By comparison, bioavailability (relative to subcutaneous injection) ofinsulin administered to rats as an inhalation liquid aerosol using asimilar endotracheal method has been reported as 37.3% (P. Colthorpe etal. Pharm. Res. 9, 764 (1992)). Absolute bioavailability of insulininhaled into rat lungs in the form of a lactose/insulin powder via a drypowder inhaler connected to an endotracheal tube has been reported as6.5% (F. Komada et al. J. Phann. Sci. 83, 863 (1994)).

Given the short systemic half life of insulin (11 minutes), and the12-24 h time scale of particle clearance from the central and upperairways, the appearance of exogenous insulin in the bloodstream severaldays following inhalation appears to indicate that large porousparticles achieve long, non-phagocytosed life-times when administered tothe deep lung. To test this hypothesis, the lungs of rats were lavagedboth inunediately following inhalation of the porous and nonporousinsulin particles, and 48 h after inhalation.

In the case of nonporous particles, 30%±3% of phagocytic cells containedparticles immediately following inhalation, and 39% ±5% containedparticles 48 h after inhalation. By contrast only 8%±2% of phagocyticcells contained large porous particles right after inhalation, and12.5%±13.5% contained particles 48 h after inhalation. In the smallnonporous particle case, 17.5%±1.5% of the phagocytic cell populationcontained 3 or more particles 48 h after inhalation. compared to 4%±1%in the case of the large nonporous particles. Inflammatory response wasalso elevated in the small nonporous particle case; neutrophilsrepresented 34%±12% of the phagocytic cell population 48 h followinginhalation of the small nonporous particles, compared to 8.5%±3.5% inthe large porous particle case (alveolar macrophages represented 100% ofphagocytic cells immediately following inhalation). These resultssupport in vitro experimental data appearing elsewhere that showphagocytosis of particles diminishes precipitously as particle diameterincreases beyond 3μm (H. Kawaguchi, et al. Biomaterials 7, 61 (1986). L.J. Krenis, and B. Strauss, Proc. Soc. Exp. Med. 107, 748 (1961). S.Rudt, and R. H. Muller, J. Contr. Rel. 22, 263 (1992)).

EXAMPLE 8 In Vivo Aerosolization of PLGA Porous Particles IncludingTestosterone

A second model drug, testosterone, was encapsulated in porous particlesof two different mean geometric diameters (10.1 μm and 20.4 μm) tofurther determine whether increased bioavailability correlates withincreasing size of porous particles. An identical mass of powder wasadministered to rats as an inhalation aerosol or as a subcutaneousinjection (controls). Serum testosterone concentrations were monitoredas a function of time following inhalation or injection (FIGS. 7 and 8).Blood levels of testosterone remain well above background levels(p<0.05) for between 12 and 24 h, even though the systemic half-life oftestosterone is between 10 and 20 minutes. Testosterone bioavailabilityrelative to subcutaneous injection is 177% for the 20.4 μm diameterparticles (FIG. 7) and 53% for the 10.1 μm diameter porous particles(FIG. 8).

The increase in testosterone bioavailability with increasing size ofporous particles is especially notable given that the mean diameter ofthe 20.4 μm particles is approximately ten times larger than that ofnonporous conventional therapeutic particles (D. Ganderton, J.Biopharmaceutical Sciences 3, 101 (1992). The relatively short timescale of testosterone release observed both for the inhalation andsubcutaneous controls is near the several hour in vitro time scale ofrelease reported elsewhere for 50:50 PLGA microparticles of similar sizeencapsulating a therapeutic substance (bupivacaine) of similar molecularweight and lipophilicity (P.-Le Corre et al. Int. J. Pharm. 107, 41(1994)).

By making particles with high porosity, relatively large particles(i.e., those possessing the same aerodynamic diameter as smaller,nonporous particles) can enter the lungs, since it is particle mass thatdictates location of aerosol deposition in the lungs. The increasedaerosolization efficiency of large, light particles lowers theprobability of deposition losses prior to particle entry into theairways, thereby increasing the systemic bioavailability of an inhaleddrug.

What is claimed is:
 1. A particulate system for delivery to thepulmonary system comprising particles which comprise a biodegradablematerial and a therapeutic, prophylactic or diagnostic agent, have a tapdensity of less than 0.4 g/cm³ and a mass mean diameter between about 5μm and about 30 μm.
 2. The particulate system of claim 1 wherein theparticles have an aerodynamic diameter less than about 5 μm.
 3. Theparticulate system of claim 1 wherein the biodegradable material is apolymer.
 4. The particulate system of claim 1 wherein the biodegradablematerial is a polysaccharide.
 5. The particulate system of claim 1wherein the agent is a hormone.
 6. The particulate system of claim 5wherein the agent is insulin.
 7. The particulate system of claim 1wherein the agent is an antibody.
 8. The particulate system of claim 1wherein the agent is estradiol.
 9. The particulate system of claim 1wherein the agent is a beta agonist.
 10. The particlulate system ofclaim 1 wherein the agent is salicylate.
 11. The particulate system ofclaim 1 wherein the agent is leuprolide.
 12. The particulate system ofclaim 1 wherein the agent is somatostin.
 13. A particulate system fordelivery to the pulmonary system comprising particles which comprise abiodegradable material and a therapeutic, prophylactic or diagnosticagent, and wherein at least 90% of the particles have a tap density ofless than 0.4 g/cm³, and a mass mean particle diameter between about 5μm and about 30 μm.
 14. A particulate system for delivery to thepulmonary system comprising particles which comprise a biodegradablematerial and a therapeutic, prophylactic or diagnostic agent, have a tapdensity less than 0.1 g/cm³ and a mass mean diameter between about 5 μmand about 30 μm.
 15. A particulate system for delivery to the pulmonarysystem comprising particles which comprise a biodegradable material anda therapeutic, prophylactic or diagnostic agent, have an envelope massdensity of less than 0.4 g/cm³, and a particle diameter between about 5μm and about 30 μm.
 16. A method for delivering particles to thepulmonary system comprising administering to the respiratory tract of apatient in need of treatment, diagnosis or prophylaxis, an effectiveamount of particles which comprise a biodegradable material and atherapeutic, prophylactic or diagnostic agent, wherein the particleshave a tap density of less than 0.4 g/cm³ and a mass mean diameterbetween about 5 μm and about 30 μm.
 17. The method of claim 16 whereinthe particles have an aerodynamic diameter less than about 5 μm.
 18. Themethod of claim 16 wherein the biodegradable material is a polymer. 19.The method of claim 16 wherein the biodegradable material is apolysaccharide.
 20. The method of claim 16 wherein the agent is ahormone.
 21. The method of claim 20 wherein the agent is insulin. 22.The method of claim 16 wherein the agent is an antibody.
 23. Theparticulate system of claim 16 wherein the agent is estradiol.
 24. Themethod of claim 16 wherein the agent is a beta agonist.
 25. The methodof claim 16 wherein the agent is salicylate.
 26. The method of claim 16wherein the agent is leuprolide.
 27. The method of claim 16 wherein theagent is somatostin.
 28. The method of claim 16 wherein delivery is tothe deep lung.
 29. The method of claim 16 wherein delivery is to theairways.
 30. The method of claim 16 wherein the particles areadministered through a dry powder inhaler.
 31. The method of claim 16wherein the particles biodegrade within the pulmonary system therebyreleasing the agent at a controlled rate.
 32. The method of claim 16wherein the particles are administered in combination with apharmaceutically acceptable carrier.
 33. A method for deliveringparticles to the pulmonary system comprising administering to therespiratory system of a patient in need of treatment, prophylaxis ordiagnosis an effective amount of particles which comprise abiodegradable material and a therapeutic, prophylactic or diagnosticagent, wherein at least 90% of the particles have a tap density of lessthan 0.4 g/cm³, and a mass mean diameter between about 5 μm and about 30μm.
 34. A method for delivering particles to the pulmonary systemcomprising administering to the respiratory system of a patient in needof treatment, prophylaxis or diagnosis an effective amount of particleswhich comprise a biodegradable material and a therapeutic, prophylacticor diagnostic agent, wherein the particles have a tap density of lessthan 0.1 g/cm³, and a mass mean diameter between about 5 μm and about 30μm.
 35. A method for delivering particles to the pulmonary systemcomprising administering to the respiratory system of a patient in needof treatment, prophylaxis or diagnosis an effective amount of particleswhich comprise a biodegradable material and a therapeutic, prophylacticor diagnostic agent, wherein the particles have an envelope mass densityof less than 0.4 g/cm³, and a particle diameter between about 5 μm andabout 30 μm.